High-affinity, low molecular-mass displacers for ion-exchange chromotography

ABSTRACT

A method for separating one or more components of a biomolecule mixture by means of an ion exchange chromatographic system operated in the displacement mode includes sequentially perfusing the system with a first solution including the biomolecule mixture, and a second solution including a displacer having a structure selected from formula I and formula II:

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of Ser. No. 10/377,080, filed Feb. 28,2003, now U.S. Pat. No. 6,929,747, which claims priority from U.S.Provisional Application Ser. No. 60/360,288, filed Feb. 28, 2002, theentire contents of which are incorporated herein by reference.

FEDERALLY SPONSORED RESEARCH

This invention was made with support from NIH Grant GM 47372-O4A2.

FIELD OF THE INVENTION

The invention relates to low molecular weight displacers for separationof biomolecule mixtures by ion exchange chromatography.

BACKGROUND OF THE INVENTION

Interest in biomolecules including proteins and oligonucleotides hasexploded in recent years, as evidenced by the rise of the biotechnologyindustry. These materials are being intensively studied in a variety ofways and for many purposes, ranging from identification of individualcomponents of complex biological mixtures through determination ofQuantitative Structure Efficacy Relationships (QSER) to identificationof biopharmaceutical drug candidates.

While supplies of raw materials are relatively abundant, an ongoingproblem encountered in this work is separation and/or purification ofthese materials. Chromatographic methods are usually the method ofchoice, but the separations can be very challenging. Natural sources ofbiomolecules yield complex mixtures of materials with relatively lowconcentration of the desired product. Synthetic techniques produce crudeproducts that may be contaminated by closely related impurities, forexample, proteins differing in composition by only a few (or even asingle) amino acid. Therefore, development of efficient andcost-effective methods for separation and purification of biomoleculesis increasingly important.

Displacement chromatography can be used to perform such difficultseparations in an efficient and cost effective manner. A particularlyattractive feature of displacement chromatography is the ability toconcentrate components of a mixture during the separation. Because ofthis combination of features, the technique is especially appropriatefor industrial process-scale chromatography.

The key operational characteristic that distinguishes displacement fromstep elusion or step gradient chromatography is the use of a displacercompound that has greater affinity for these stationary phase than thedesired product. The displacer competes for absorption sites on thestationary phase, causing the feed components to exit the column asadjacent “square wave” zones of highly concentrated pure material, inthe order of increasing affinity of absorption. An important distinctionbetween displacement and step gradient chromatography is that thedisplacer front always remains behind the adjacent feed zones in thedisplacement train, while, desorbents, for example, salts and organicmodifiers, move through the feed zones. The implications of thisdistinction are quite significant in that displacement chromatographycan concentrate and purify components from mixtures having lowseparation factors, while in the case of desorption chromatography,relatively large separation factors are generally required to givesatisfactory resolution.

A limited number of materials for use as displacers in chromatographicsystems have been described in the patent and scientific literature.These include large polyelectrolytes as displacers for separation ofproteins in ion exchange systems. In addition, several types oflow-molecular weight displacers for ion exchange have also beendescribed. (See, for example, U.S. Pat. Nos. 5,478,924; 5,606,033 and6,239,262). Low-molecular weight displacers have significant operationaladvantages as compared to large polyelectrolyte displacers. First andforemost, if there is any overlap between the displacer and the proteinof interest, these low-molecular weight materials can be readilyseparated from the purified protein during post-displacement processingusing standard size-based purification methods, for example, sizeexclusion chromatography and/or ultra filtration. This advantage isparticularly important for meeting FDA standards for validatingdisplacement chromatic graphic bioprocesses. The salt dependentadsorption behavior of these low-molecular weight displacers greatlyfacilitates column regeneration. Finally, the use of low-molecularweight displacers enables the operation of displacements in theselective displacement mode which can result in elution of the weaklyretained proteins in the induced salt gradient, displacement of thebioproduct of interest and closely related impurities, and desorption ofthe more strongly retained impurities after breakthrough of thedisplacer front.

However, the major obstacle to the implementation of displacementchromatography has been a lack of displacer molecules for particularseparations. Therefore, there is a continuing need for additionaldisplacer compounds that have high affinity for the stationary phase ofion exchange chromatographic systems, as well as chemically selectivedisplacers for specific separations.

SUMMARY OF THE INVENTION

It has been unexpectedly discovered that displacers having the structureof formula I or formula II have high affinity for ionic exchangestationary phase materials.

Because of this high affinity, these displacers can be very effectivelyused for purification or separation of biomolecules such as proteins andoligonucleotides.

Accordingly, the present invention relates to a method for separatingone or more components of a biomolecule mixture by means of an ionexchange chromatographic system operated in the displacement mode. Theseparation is performed by sequentially perfusing the system with afirst solution including the biomolecule mixture, and a second solutionincluding a displacer having a structure selected from formula I andformula II.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a ranking of dynamic affinities of displacers on Source 15Qstationary phase.

FIG. 2 shows displacement separation of a mixture of β-lactoglobulin Aand β-lactoglobulin using PGPSNa₃ as a displacer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for separating one or morecomponents of a biomolecule mixture by means of an ion exchangechromatographic system operated in the displacement mode. The methodincludes sequentially perfusing the system with a first solutionincluding the biomolecule mixture, and a second solution including adisplacer. The displacer is an ion having a structure selected fromformula I and formula II:

wherein Ar is phenyl, naphthalenyl, phenanthrenyl, anthracenyl,perylenyl, pyrenyl, fluorenyl, triazolyl, carbazolyl, indolyl,benzocarbazolyl, acridinyl, phenothiazinyl, or thianthrenyl;

R¹, R², and R³ are independently alkyl, aryl, heteroalkyl, alkylaryl,oxaalkyl, oxaaryl, oxaalkylaryl, substituted alkyl, substituted aryl,substituted alkylaryl, substituted oxaalkyl, substituted oxaaryl,substituted oxaalkylaryl or a direct bond; and at least one of R¹, R²,and R³ is other than a direct bond;

X¹, X², and X³ are independently hydrogen, X⁵ or X⁶; and at least one ofX¹, X², and X³ is other than hydrogen

X⁴ is sulfonate, sulfate, phosphate, carboxylate, tertiary amino orquaternary amino;

X⁵ is sulfonate, sulfate, phosphate, or carboxylate;

X⁶ is tertiary amino or quaternary amino;

Y¹, Y², and Y³ are independently O, S or a direct bond; and Z is CH orN; and

n is an integer from 1 to 6.

In formula I, the Ar group may be mono-, di-, or tri-substituted asspecified above, that is, substituted with one or more of the groupsY¹R¹X¹, Y²R²X², and Y³R³X³. In formula II, the Ar group may besubstituted with up to six aryl or heteroaryl groups, each having anionic substitutent selected from sulfonate, sulfate, phosphate,carboxylate, tertiary amino and quaternary amino.

The counterion(s) for the displacer ion of formula is(are) not critical,and may be any appropriately charged ion. For example, a preferredcounterion for negatively charged displacers of formula I or II is Na⁺.

In one embodiment, the displacers of formula I are trisubstituted phenylor triazolyl derivatives. Preferred groups for R¹, R², and R³ are alkyl,aryl, and oxaalkyl. Examples of displacers of formula I are:

In another embodiment, displacers of formula II are phenyl derivatives,that is, Ar is phenyl. Preferred substituents are also phenyl. Aparticularly preferred displacer is:

Components of the biomolecule mixture include at least one biomolecule,for example, biological macromolecules, such as proteins oroligonucleotides, or other naturally occurring molecules such aspeptides, viruses or vaccines. Proteins differ from peptides inmolecular weight. In the context of the present invention, peptides aredefined as having molecular weight of less than 10,000 Daltons, whileproteins have molecular weight of greater than 10,000 Daltons.

The biomolecule mixture to be separated typically contains one type ofbiomolecule, such as a mixture of closely related proteins oroligonucleotides. If desired, mixtures containing complex mixtures ofdifferent types of compounds may also be separated.

Separation of a component of the mixture yields that component in a formcontaining fewer impurities than in the starting mixture. One aspect ofseparating a component of a biomolecule mixture is purifying thatcomponent, and, in one embodiment, the method of the present inventionmay be used to purify a biomolecule. In this embodiment, the separationtypically yields a product of at least 90% purity, and the purity may beas high as 99%. The method is particularly useful when the desiredbiomolecule is contaminated with a closely related impurity which isdifficult to remove by conventional methods. However, the impurity(ies)may be any unwanted contaminant of the biomolecule to be purified,including low molecular weight materials.

Conventional hardware systems may be used for the separation, includingcomponents such as a column, pump, and detector, with minormodifications to enable the sequential perfusion of the column with thecarrier, feed, displacer, and regenerant solutions (Shukla A. A. andCramer S. M; Bioseparations by Displacement Chromatography, in Ahuja(ed.); Handbook of Bioseparations; Academic Press; accepted forpublication; Frenz, J. and Horvath, Cs., High PerformanceChromatography-Advances and Perspectives, in: Cs. Horvath (ed.), HighPerformance Displacement Chromatography; Vol. 5; Academic Press (1988)).The IEX system also includes a stationary phase and a mobile phase, asin conventional systems.

Suitable matrix materials for the stationary phase include those basedon agarose, polymethylmethacrylate, hydrophilizedpolystyrene-divinylbenzene, dextran, or cellulose. In chromatographicsystems for separating negatively charged species, the matrix istypically functionalized with a cationic group, such as a quaternaryamine group. Negatively charged displacers, that is, those bearingsulfonate, sulfate, phosphate, or carboxylate functionality, are usedwith a cationic stationary phase. In systems, for separating positivelycharged species, the matrix is typically functionalized with an anionicgroup, such as a sulfonate group. Positively charged displacers, thatis, those bearing tertiary amino or quaternary amino functionality, areused with an anionic stationary phase.

The mobile phase is typically also a conventional one, usually abuffered aqueous solution. The mobile phase is used as the carriersolution, as the solvent for the biomolecule mixture in the feedsolution, and as the solvent for the displacer in the displacersolution. The regenerant solution also includes the mobile phase, andmay have additional solvents to facilitate removal of the displacer fromthe system.

In performing a separation according to the method of the presentinvention, the IEX system is operated in displacement mode. Displacementmode differs from conventional elution or step gradient modes primarilyin that a displacer is passed through the column behind or after thesample. When operated in displacement mode, the system is sequentiallyperfused with a first or feed solution including the biomoleculemixture, and a second or displacer solution including the displacer. Ifdesired, the system may be equilibrated with the carrier solution beforebeginning perfusion of the feed solution. After the separation, it isusually desirable to regenerate the column by removing the displacerfrom the system. This may be done by passing a regenerant solutionthrough the column. Displacers useful in the methods of the presentinvention are typically removed from the column after washing with fromfive to twenty column volumes of the regenerant solution, although, insome cases, use of much more or much less solution may be desirable. Thecomposition of the regenerant solution is typically optimized for aparticular displacement, and may contain organic solvents in addition tothe mobile phase aqueous solution.

In the context of the present invention, alkyl is intended to includelinear, branched, or cyclic hydrocarbon structures and combinationsthereof. Lower alkyl refers to alkyl groups of from 1 to 4 carbon atoms.Lower alkyl groups include methyl, ethyl, n-propyl, isopropyl, and n-,s- and t- butyl. Preferred alkyl groups are those of C₂₀ or below.Cycloalkyl is a subset of alkyl and includes cyclic hydrocarbon groupsof from 3 to 8 carbon atoms. Examples of cycloalkyl groups includecyclopropyl, cyclobutyl, cyclopentyl, and norbornyl

Alkoxy or alkoxyl refers to groups of from 1 to 8 carbon atoms of astraight, branched, cyclic configuration and combinations thereofattached to the parent structure through an oxygen. Examples includemethoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, and cyclohexyloxy.Lower alkoxy refers to groups containing one to four carbons.

Acyl refers to groups of from 1 to 8 carbon atoms of a straight,branched, cyclic configuration, saturated, unsaturated and aromatic andcombinations thereof, attached to the parent structure through acarbonyl functionality. One or more carbons in the acyl residue may bereplaced by nitrogen, oxygen or sulfur as long as the point ofattachment to the parent remains at the carbonyl. Examples includeacetyl, benzoyl, propionyl, isobutyryl, t-butoxycarbonyl, andbenzyloxycarbonyl. Lower-acyl refers to groups containing one to fourcarbons.

Aryl and heteroaryl mean a 5- or 6-membered aromatic or heteroaromaticring containing 0–3 heteroatoms selected from nitrogen, oxygen orsulfur; a bicyclic 9- or 10-membered aromatic or heteroaromatic ringsystem containing 0–3 heteroatoms selected from Nitrogen, oxygen orsulfur; or a tricyclic 13- or 14-membered aromatic or heteroaromaticring system containing 0–3 heteroatoms selected from Nitrogen, oxygen orsulfur. Each of these rings is optionally substituted with 1–3 loweralkyl, substituted alkyl, substituted alkynyl, carbonyl, nitro, halogen,haloalkyl, hydroxy, alkoxy, OCH(COOH)₂, cyano, primary amino, secondaryamino, acylamino, phenyl, benzyl, phenoxy, benzyloxy, heteroaryl, orheteroaryloxy; each of said phenyl, benzyl, phenoxy, benzyloxy,heteroaryl, and heteroaryloxy is optionally substituted with 1–3substituents selected from lower alkyl, alkenyl, alkynyl, halogen,hydroxy, haloalkyl, alkoxy, cyano, phenyl, benzyl, benzyloxy,carboxamido, heteroaryl, heteroaryloxy, nitro or —NRR (wherein R isindependently H, lower alkyl or cycloalkyl, and —RR may be fused to forma cyclic ring with nitrogen). The aromatic 6- to 14-membered carbocyclicrings include, for example, benzene, naphthalene, indane, tetralin, andfluorene; and the 5- to 10-membered aromatic heterocyclic rings include,e.g., imidazole, pyridine, indole, thiophene, benzopyranone, thiazole,furan, benzimidazole, quinoline, isoquinoline, quinoxaline, pyrimidine,pyrazine, tetrazole and pyrazole.

Alkylaryl means an alkyl residue attached to an aryl ring. Examples arebenzyl and phenethyl. Heteroarylalkyl means an alkyl residue attached toa heteroaryl ring. Examples include pyridinylmethyl andpyrimidinylethyl.

Heterocycle means a cycloalkyl or aryl residue in which one to two ofthe carbons is replaced by a heteroatom such as oxygen, nitrogen orsulfur. Examples of heterocycles that fall within the scope of theinvention include pyrrolidine, pyrazole, pyrrole, indole, quinoline,isoquinoline, tetrahydroisoquinoline, benzofuran, benzodioxan,benzodioxole (commonly referred to as methylenedioxyphenyl, whenoccurring as a substituent), tetrazole, morpholine, thiazole, pyridine,pyridazine, pyrimidine, thiophene, furan, oxazole, oxazoline, isoxazole,dioxane, and tetrahydrofuran.

Substituted alkyl, aryl, cycloalkyl, or heterocyclyl refer to alkyl,aryl, cycloalkyl, or heterocyclyl wherein up to three H atoms in eachresidue are replaced with halogen, haloalkyl, hydroxy, lower alkoxy,carboxy, carboxalkoxy, carboxamido, cyano, carbonyl, nitro, primaryamino, secondary amino, alkylthio, sulfoxide, sulfone, acylamino,acyloxy, amidino, phenyl, benzyl, heteroaryl, phenoxy, benzyloxy,heteroaryloxy, or substituted phenyl, benzyl, heteroaryl, phenoxy,benzyloxy, or heteroaryloxy.

Haloalkyl refers to an alkyl residue, wherein one or more H atoms arereplaced by halogen atoms; the term haloalkyl includes perhaloalkyl.Examples of haloalkyl groups that fall within the scope of the inventioninclude CH₂F, CHF₂, and CF₃.

Oxaalkyl refers to an alkyl residue in which one or more carbons havebeen replaced by oxygen and attached to the parent structure through anoxygen. Examples include methoxypropoxy, 3,6,9-trioxadecyl and the like.The term oxaalkyl is intended as it is understood in the art [see Namingand Indexing of Chemical Substances for Chemical Abstracts, published bythe American Chemical Society, ¶196, but without the restriction of¶127(a)], i.e. it refers to compounds in which the oxygen is bonded viaa single bond to its adjacent atoms (forming ether bonds); it does notrefer to doubly bonded oxygen, as would be found in carbonyl groups.Similarly, thiaalkyl and azaalkyl refer to alkyl residues in which oneor more carbons has been replaced by sulfur or nitrogen, respectively.Examples include ethylaminoethyl and methylthiopropyl.

Anionic displacers of formula I may be synthesized by the methodsillustrated in Schemes 1 and 2.

Anionic displacers of formula II, where n=6, may be synthesized by themethod illustrated in Scheme 3.

Where n<6, the displacers may be prepared by functionalizingcommercially available starting materials, such as biphenyl orterphenyl.

Cationic displacers may be synthesized by similar methods. While Schemes1, 2 and 3 show preparation of anionic displacers having sulfonate ionicsubstituents, cationic displacers having amino substituents are readilysynthesized by modification of the functionalization step(s). Methodsfor functionalizing an aromatic ring with a tertiary or quaternary amineare known in the art.

The following examples illustrate preparation of displacers having thestructure of formulas I and II, use of these displacers to separateclosely related proteins in an anionic IEX system, and the results ofscreening experiments that indicate that the displacers are alsoeffective for separation of oligonucleotides.

EXAMPLES Example 1 Synthesis of Displacers

IC—PS Na₃: To a suspension of sodium hydride of 95% purity (1.80 g, 0.07mol) in anhydrous DMSO (50 mL) was added cyanuric acid (2.64 g, 0.02mol) in anhydrous DMSO (50 mL) dropwise at room temperature under anargon atmosphere. After the addition of cyanuric acid(1,3,5-triazine-2,4,6-triol), the reaction mixture was stirred at 80° C.for 1 hr. The temperature of the reaction mixture was lowered to 50° C.and then 1,3-propane sultone (10 g, 0.08 mol) in anhydrous DMSO (30 mL)was added dropwise to the suspension. At this temperature, the reactionmixture was stirred for 1 day. As the reaction proceeded, the suspensionbecame yellowish in color. Finally the reaction mixture was stirred at80° C. for one hour. After cooling to room temperature, the reactionmixture was filtered. THF (300 mL) was added to the resulting solutionwhile stirring. The resultant white precipitate was filtered, and driedin a vacuum oven at 60° C. For further purification, the white productwas placed in 400 mL of methanol and the suspension was boiled until thesuspension was concentrated to 200 mL. After cooling to roomtemperature, the white solid was filtered, and dried in a vacuum oven at110° C. for 3 days to afford 7.9 g of IC—PSNa₃ in a yield of 70%. ¹H NMR(DMSO-d₆): δ 3.77 (t, —NCH ₂CH₂CH₂SO₃Na, 6H), 2.47 (t, —NCH₂CH₂CH₂SO₃Na, 6H), 1.87 (quintet, —NCH₂CH ₂CH₂SO₃Na, 6H). ¹³C NMR (DMSO-d₆): δ149.01 (—N—C═O), 48.94 (—NCH₂CH₂CH₂SO₃Na), 41.82 (—NCH₂CH₂ CH₂SO₃Na),23.73 (—NCH₂ CH₂CH₂SO₃Na). IR (KBr): 1681 (vs, C═O), 1472 (s), 1206 (s),1058 (s), 764, 613, 531 cm⁻¹.

PG-PSNa₃: To a suspension of sodium hydride of 95% purity (1.07 g, 0.04mol) in anhydrous HMPA (40 mL) was added phloroglucinol (1.53 g, 0.01mol) dropwise at room temperature under an argon atmosphere. Thereaction mixture was stirred at room temperature for 5 hr. 1,3-Propanesultone (5.18 g, 0.04) in HMPA (20 mL) was added dropwise to thesuspension and the reaction mixture was stirred at room temperature for12 hr. The temperature of the reaction mixture was raised to 80° C. andthe mixture was stirred for 12 hr. After cooling to room temperature,the reaction mixture was filtered. The solution was poured into amixture of ethanol/acetone (100 mL/400 mL). The resultant precipitatewas filtered, washed with ethanol and acetone, and dried in a vacuumoven at 60° C. To purify further, the product was placed in 300 mL ofmethanol and the suspension was boiled and concentrated to 150 mL. Aftercooling to room temperature, the white solid was filtered, and dried ina vacuum oven at 100° C. for 2 days to afford 5.0 g of PG-PSNa₃ in ayield of 74%. ¹H NMR (DMSO-d₆): δ 6.04 (s, aromatic protons, 3H), 3.98(t, —OCH ₂CH₂CH₂SO₃Na, 6H), 2.55 (t, —OCH₂CH₂ CH₂SO₃Na, 6H), 1.97(quintet, —OCH₂CH ₂CH₂SO₃Na, 6H). ¹³C NMR (DMSO-d₆): δ 160.47 (aromaticC—OCH₂—), 93.75 (aromatic C—H), 66.66 (—OCH₂CH₂CH₂SO₃Na), 47.92(—OCH₂CH₂ CH₂SO₃Na), 25.18 (—OCH₂ CH₂CH₂SO₃Na).

TA-PSNa₃: To a suspension of sodium hydride of 95% purity (1.82 g, 0.072mol) in anhydrous DMSO (200 mL) was added 3-hydroxy-1-propanesulfonicacid sodium salt (11.50 g, 0.056 mol) in three portions at roomtemperature under an argon atmosphere. The reaction mixture was stirredat room temperature for 8 hr. Cyanuric chloride(2,4,6-trichloro-1,3,5-triazine, 3.000 g, 0.016 mol) was added to thesuspension in three portions and then the reaction mixture was stirredat room temperature for one day. As the reaction proceeded, the reactionmixture became yellowish in color. The reaction mixture was filtered toremove some salt. The resulting solution was poured into acetone (300mL) with stirring. The white precipitate was filtered, and dried in avacuum oven at 60° C. For further purification, the product wasrecrystallized from water/ethanol (1/4). The white solid was filtered,and dried in a vacuum oven at 110° C. for 3 days to afford 4.0 g ofTA-PSNa₃ in a yield of 48%. ¹H NMR (DMSO-d₆): δ 4.20 (t, —OCH₂CH₂CH₂SO₃Na, 6H), 2.53 (t, —OCH₂CH₂ CH₂SO₃Na, 6H), 1.91 (quintet, —OCH₂CH₂CH₂SO₃Na, 6H). ¹³C NMR (DMSO-d₆): δ 172.01 (—N═C—O—), 64.37 (—OCH₂CH₂CH₂SO₃Na), 47.91 (—OCH₂CH₂ CH₂SO₃Na), 24.98 (—OCH₂ CH₂CH₂SO₃Na).

1,3,5-Tris(2-hydroxyethoxy)benzene: A mixture of phloroglucinol (12.60g, 0.10 mol), ethylene carbonate (35 g, 0.39 mol), andtetraethylammonium bromide (8 g, 0.04 mol) in DMF (20 mL) was heated at150° C. for 14 hr. After evaporating the solvent, the residue waspurified on a silica gel chromatography column using a mixture ofmethylene chloride and acetonitrile: 1/1, 1/2, 1/3 and 1/4 as eluents togive 9.6 g of 1,3,5-tris(2-hydroxyethoxy)benzene (37% yield). Theproduct was purified further by recrystallization from cold methylenechloride and THF to afford 5.5 g (21%). ¹H NMR (DMSO-d₆): δ 6.08 (s,aromatic protons, 3H), 4.83 (s, OH, 3H), 3.92 (t, —OCH ₂—, 6H), 3.69 (m,—CH ₂OH, 8H). ¹³C NMR (DMSO-d₆): δ 160.54 (aromatic C—O—),93.81(aromatic C—H), 69.53 (—CH ₂O—), 59.55 (—CH₂OH). FT-IR (KBr, m⁻¹):3255 (OH), 2947, 2371, 1596, 1462, 1377, 1167, 1071, 1024, 902, 745.

1,3,5-Tris(3-tosyloxy-1-oxapropyl)benzene: To a solution of 1(3.0 g,12.00 mmol) in pyridine (30 mL) was added p-toluensulfonylchloride(11.40 g, 60 mmol) in pyridine (30 mL) dropwise at 0° C. under nitrogen.The reaction mixture was stirred at 0–20° C. for 1 day. The resultingsalt was filtered and then two thirds of the pyridine was distilledunder vacuum at room temperature. Into the resulting solution was pouredice/water with stirring. The precipitate was filtered and dried undervacuum at room temperature, overnight. The product was dissolved inchloroform, dried over magnesium sulfate, and the solid was filtered.After one half the solvent in the filtrate was removed on a rotaryevaporator, methanol was added to the solution until the volume ratio ofboth solvents reached approximately one to one. The mixture was allowedto stand overnight in the refrigerator to initiate precipitation. Theresulting solid was filtered and dried in a vacuum oven at 60° C.overnight to give 6.5 g of 1,3,5-tris(3-tosyloxy-1-oxapropyl)benzene(75% yield). ¹H NMR (DMSO-d₆): δ 7.81 (d, 6H), 7.47 (d, 6H); protons onthe phenyl moiety of the tosyl group, 5.99 (s, protons on the phenylmoiety of the phloroglucinoxy group, 3H), 4.31 (t, PhOCH ₂CH₂O—, 6H),4.09 (t, PhOCH ₂CH₂O—, 6H), 2.40 (s, —CH₃, 9H).

PG(EO1)-PSNa₃: To a suspension of NaH (0.31 g, 12.40 mmol) in anhydrousDMSO (20 mL), 1,3,5-tris(2-hydroxyethoxy)benzene (0.80 g, 3.10 mmol) wasadded slowly at room temperature under nitrogen. The mixture was stirredfor 4 hours. 1,3-Propane sultone (1.90 g, 15.49 mmol) was added to thesuspension at room temperature and then the reaction mixture was heatedat 80° C. for 1 day. The reaction mixture was cooled to room temperatureand poured into isopropanol. The resulting precipitate was filtered anddried under vacuum at 60° C. overnight. The product was purified furtherby fractional precipitation from DMSO by adding increasing amounts ofethanol. The first precipitate was filtered and the second was collectedtaken from ethanol and dried in a vacuum oven at 60° C. overnight.Finally, the product was purified further by precipitation fromDMSO/ethanol/water to give 0.7 g of PG(EO1)-PSNa₃ (51% yield). m.p.=253°C. (by DSC). ¹H NMR (DMSO-d₆): δ 6.11(s, aromatic H, 3H), 4.03 (t, PhOCH₂—, 6H), 3.65 (t, —OCH₂CH ₂OCH₂—, 6H), 3.49 (t, —OCH ₂CH₂CH₂—), 2.48 (t,—CH₂SO₃Na, 6H), 1.81 (quintet, —OCH₂CH ₂CH₂—, 6H). ¹³C NMR (DMSO-d₆): δ160.34 (aromatic C—OH), 93.86 (aromatic C—H), 69.65 (PhOCH₂—), 68.47(—OCH₂ CH₂OCH₂—), 67.18 (—OCH₂CH₂CH₂—), 48.24 (—CH₂SO₃Na), 25.60 (—OCH₂CH₂CH₂—).

PG(EO1)-PhSO₃Na: 4-Hydroxybenzenesulfonic acid sodium salt dihydrate(2.18 g, 9.40 mmol), sodium hydroxide (0.36 g, 8.95 mmol) in DMSO (25mL) and HMPA (10 mL) were placed in a 2-neck-round bottom flask equippedwith a Dean-Stark trap and condenser under nitrogen. The reactionmixture was heated at 130–140° C. while adding and distilling tolueneseveral times for 1 day to generate anhydrous phenoxide ions. Themixture was cooled to room temperature. To the suspension was added1,3,5-tris(3-tosyloxy-1-oxapropyl)benzene (1.50 g, 2.08 mmol) and thenthe temperature was raised to 80° C. and stirred for 1 day. Aftercooling to room temperature, the reaction mixture was poured intoisopropyl alcohol with stirring. The resulting precipitate was filteredand dried in a vacuum oven at 60° C. overnight. The product was purifiedfurther by reprecipitation from water and isopropyl alcohol, and theobtained product was dried in a vacuum oven at 60° C. for 2 days to give1.50 g (91% yield) of PG(EO1)-PhSO₃Na. m.p.=333° C. (on DSC). ¹H NMR(DMSO-d₆): δ 7.55(d, ortho-protons to the sulfonyl group, 6H), 6.92 (d,meta-proton to the sulfonyl group, 6H), 6.23 (s, protons on the phenylring of phloroglucinoxy group, 3H), 4.29 (br.s, —OCH ₂CH ₂O—, 12H). ¹³CNMR (DMSO-d₆): δ 160.24 (para-carbon to the sulfonyl group), 158.36(aromatic C—O of the phloroglucinoxy group), 141.11(C—SO₃Na), 127.14(ortho-carbon to the sulfonyl group), 113.37 (meta-carbon to thesulfonyl group), 94.15 (aromatic C—H of the phloroglucinoxy group),66.43, 66.37 (—OCH₂ CH₂O—).

TA-PhSO₃Na: 4-Hydroxybenzenesulfonic acid sodium salt dihydrate (4.00 g,16.90 mmol) and sodium hydroxide (0.67 g, 16.46 mmol) in DMSO (60 mL)were placed in a 2-neck-round bottom flask equipped with a Dean-Starktrap and a condenser under nitrogen. The reaction mixture was heated at130–140° C. while adding and distilling toluene several times forone-day to generate anhydrous phenoxide ion. The mixture was cooled toroom temperature. To the suspension was added cyanuric chloride (0.70 g,3.79 mmol) slowly with stirring at room temperature for one-day. Theresulting precipitate was filtered and dried in a vacuum oven at 60° C.overnight. The product was precipitated from ethanol and the obtainedproduct was filtered and dried in a vacuum oven at 60° C. for one-day.The white solid was purified further by reprecipitation from the water,DMSO ethanol mixture to give 1.40 g (56%) of TA-PhSO₃Na m.p.=440° C. (byDSC). ¹H NMR (DMSO-d₆): δ 7.68(d, ortho-proton to the sulfonyl group,6H), 7.22(d, meta-proton to the sulfonyl group, 6H). ¹³C NMR (DMSO-d₆):δ 173.06 (carbon atom of the triazine ring), 151.32 (para-carbon atom tothe sulfonyl group), 145.98 (C—SO₃Na), 127.03(ortho-carbon atom to thesulfonyl group), 120.75 (meta-proton to the sulfonyl group).

1,4 Hexaphenylbenzene: A mixture of tetraphenylcyclopentadienone (8.0 g,21.0 mmol), diphenylacetylene (8.0 g, 43.0 mmol), and benzophenone (40g) was heated at 320° C. for one-hour in a one-neck round bottom flaskattached to an air condenser. The reaction mixture was cooled to roomtemperature to give a crystalline precipitate which was filtered, washedwith benzene and dried in a vacuum oven at 80° C. overnight. The productwas purified further by recrystallization from diphenylether, filteredand washed with hot benzene to give 8.1 g of 1,4 hexaphenylbenzene(72%). ¹H NMR (THF-d₈): δ 6.81−6.75 (m, C—H, 30H). ¹³C NMR (THF-d₈): δ141.88 (core phenyl group-C of the substituted phenyl group), 141.60(carbon atom of the core phenyl group), 132.50 (meta-carbon ofsubstituted phenyl group), 127.53 (para-carbon atom of the substitutedphenyl group), 126.09 (ortho-carbon of substituted phenyl group).

HPB—SO₃Na: To 1,4 hexaphenylbenzene (4.0 g, 7.50 mmol) was addedchlorosulfonic acid (45 mL) slowly at room temperature and the reactionmixture was stirred overnight. The solution was poured into ice/waterand the resulting precipitate was filtered and dried in a vacuum oven at60° C. to give 8.0 g (95%) of hexaphenylbenzene hexasulfonyl chloride.The product was used in the next step without further purification.

To a solution of sodium hydroxide (4.55 g, 113.80 mmol) in water (100mL) was added hexaphenylbenzene hexasulfonyl chloride (8.0 g, 7.10 mmol)at room temperature. The reaction mixture was heated at 50° C.overnight. After cooling to room temperature, the reaction mixture wasfiltered to remove a small amount of precipitate from the solution. Tothe obtained solution was added sufficient ethanol to give a white solidwith stirring. After filtering and drying, the resulting solid waspurified further by fractional precipitation from DMSO by addingincreasing amounts of ethanol to afford 5.5 g (68% yield) of HPB—SO₃Na.To purify the product further, the obtained solid was reprecipitatedfrom a mixture of DMF and water and acidified with aqueous hydrochloricacid. Finally, the desired product was obtained by precipitation from amixture of methanol and acetone. ¹H NMR (D₂O): δ 7.41 (d, ortho-protonto the sulfonyl group, 12H), 7.17 (d, meta-proton to the sulfonyl group,12H). ¹³C NMR (D₂O): δ 142.78(C—SO₃Na), 140.32 (para-carbon atom to thesulfonyl group) 139.74 (core phenyl group-C of the substituted phenylgroup), 131.91(ortho-carbon atom to the sulfonyl group), 124.40(meta-carbon atom to the sulfonyl group.

Example 2 Protein Separation by Displacement Anion ExchangeChromatography

Materials: Source 15Q Quaternary ammonium strong anion exchange columns,(15 μm, 100×4.6 mm i.d) was donated by Amersham Pharmacia Biotech(Uppsala, Sweden). ToyoPearl Super Q 650S (35 μm) and Q Sepharose HPBulk stationary phase materials were donated by TOSOH BIOSEP(Montgomeryville, Pa., USA) and Amersham Biosciences (Uppsala, Sweden),respectively. Protein-Pak Q-8HR anion exchange (100×5 mm i.d) column wasa gift from Waters Corporation (Milford, Mass., USA). TSK-Gel G3000SWXLsize exclusion column (300 m×7.8 mm i.d.) and the TSK-Gel SWXL (40×6 mmi.d) guard column were gifts from TOSOH BIOSEP (Montgomeryville, Pa.,USA). Q Sepharose HP and ToyoPearl Super Q 650S stationary phases wereslurry packed in 100×5 mm I.D. and 50×5 mm I.D. columns, respectively.Tris(hydroxymethyl)amino methane, Tris(hydroxymethyl)amino methanehydrochloride, sodium chloride, sodium nitrate, human serum albumin(HSA), bovine serum albumin (BSA), α-lactalbumin, β-lactoglobulin A andβ-lactoglobulin B from Sigma Chemical Company (St. Louis, Mo., USA).Pentaerythrityltetrabromide was purchased from Aldrich Chemical Company(Milwaukee, Wis., USA) and recrystallized from N,N-dimethylformamide(DMF) before use. N,N-Dimethylethanolamine was dried over solid NaOH anddistilled under vacuum prior to use. 2-[2-(Dimethylamino) ethoxy]ethanolwas dried and stored over molecular sieve (4A).N,N-Bis(3-aminopropyl)ethylenediamine was dried over solid NaOH,distilled under vacuum and stored over molecular sieve (4A). AnhydrousN,N-Dimethylformamide and dimethylsulfoxide (DMSO), tetrahydrofuran(THF) and ethanol from the Aldrich Chemical Company (Milwaukee, Wis.,USA) were employed without further purification. All other solvents andreagents were used without additional purification unless specified inthe procedure. The membranes for dialysis were purchased from SpectrumMedical Industries, Inc (Houston, Tex., USA) withmolecular-weight-cut-off (MWCO) of 500 and from Membrane FiltrationProducts, Inc (Seguin, Tex., USA) with MWCO of 1000.

Instrumentation and Apparatus: Linear gradients were run on a Pharmaciafast protein liquid chromatographic (FPLC) system consisting of twoP-500 pumps and a LCC-500 controller donated by Amersham Biosciences(Uppsala, Sweden). Displacement experiment was carried out using aWaters 590 HPLC pump (Waters, Milford, Mass.) connected to achromatography column via a Model C10W port valve (Valco, Houston,Tex.). The column effluent was monitored using a Waters 484 UV-Visabsorbance detector (Waters Corporation, Milford, Mass., USA) and thedata was acquired using a QuickLog (Version 1.4) chromatographyworkstation (Strawberry Tree, Inc., Sunnyvale, Calif., USA). Columneffluent fractions during displacement experiment were collected using aLKB 2212 Helirac fraction collector (LKB Bromma, Sweden). Analysis offractions collected during displacement experiments were carried outusing a Waters 600 multisolvent delivery system, a Waters 712 WISPautoinjector and a Waters 484 UV-VIS absorbance detector controlled by aMillenium chromatography software manager (Waters, Milford, Mass.). ¹Hand ¹³C NMR spectra were recorded as ppm values with a 500 MHz VarianUnity-500 spectrometer. Infrared spectra were obtained on KBr pelletswith a Perkin Elmer (Wellesley, Mass., USA) Paragon 1000 FT-IR. GC-MSspectra were obtained at injection temperatures of 250° C. and 280° C.with GC-17A and GCMS-QP5000 instruments from the Shimadzu Company(Kyoto, JAPAN). Melting points were measured on a differential scanningcalorimeter (DSC) from Perkin Elmer (Wellesley, Mass., USA) model DSC 7at a heating rate of 10° C./min under nitrogen.

Determination of Steric Mass Action (SMA) Parameters: SMA parameterswere determined according to the procedure described by Brooks et al.,AIChe J. 38, 1969 (1992). The SMA parameters were used to generatedynamic affinity plots for predicting the relative affinity of thedisplacers (Shukla, A A, et al., Ind. Eng. Chem. Res., 37, 4090 (1998)).For the displacers, linear gradient experiments were carried out atdifferent times between buffer A (50 mM Tris, pH 7.5) and buffer B (50mM Tris, pH 7.5, containing 2.5 M NaCl). Experiments were performed at0.5 ml/min for Q Sepharose HP and Source 15Q, and at 0.4 ml/min forToyoPearl Super Q-650S. Ion capacities (Λ) of the stationary phases weredetermined using a frontal method. The column was perfused with at leasttwo different concentrations of sodium nitrate solutions in theequilibration buffer (50 mM Tris, 30 mM NaCl, pH 7.5). After eachfrontal, the stationary phases were regenerated using 2M NaCl solution.The breakthrough volumes of the sodium nitrate were used to calculatethe ionic capacity of the stationary phases.

Displacer analysis: UV-VIS absorbance was used to monitor the columneffluent during linear gradient analysis of the displacers. Fordisplacer ICPSNA3, the effluent was monitored at 215 nm. For theremaining displacers, the effluent was monitored at 254 nm.

Displacement Chromatographic Separation: For the displacementexperiment, a Source 15Q (50×5 mm i.d.) the column was initiallyequilibrated with the carrier buffer 20 mM Tris+55 mM NaCl and thensequentially perfused with feed, displacer and regenerant solutions. Thefeed solution of 15.1 mg of β-lactoglobulin A and 16.7 mg ofβ-lactoglobulin B was prepared in 8 ml of carrier buffer. The proteinsolution (18 mg) was purified using 5 mM PG-PSNa₃ as the displacer.Appropriate fractions (400 μl) of column effluent were collected duringthe displacement experiments for subsequent analysis of proteins anddisplacer. The displacement experiment was carried out at a flow rate of0.2 ml/min and the effluent was monitored at 235 nm. The column wasregenerated sequentially with five column volumes of 2.5 M NaCl and 1 MNaOH solutions. Results are shown in FIG. 2.

Quantitative protein analysis of the fractions collected during thedisplacement experiment was performed by anion exchange chromatographyunder isocratic elution conditions. The proteins were analyzed using aProtein-Pak Q-8HR anion exchange (100×5 mm i.d) column with 170 mM NaClin 20 mM Tris-HCl, pH 7.5. 5 μl samples were injected at a flow rate of0.5 ml/min and the effluent was monitored at 235 nm.

Quantitative displacer analysis was performed by size exclusionchromatography using a TSK-Gel G3000SWXL (300 m×7.8 mm i.d.) in thepresence of TSK-Gel SWXL (40×6 mm i.d) guard column. 5 μl samples wereinjected at a flow arte of 1 mL/min, 50 mM phosphate+100 mM NaCl, pH 6.0as the carrier buffer. The effluent was monitored at 235 nm.

A common feature of these displacer molecules is that they all have aring structure in the core of the molecule. Table 1 shows some of thestructural characteristics of these displacers. To classify thesemolecules according to their hydrophobicity, log P (octanol/waterpartition coefficient) calculations were performed using MOE software(Chemical Computing Group, Inc., Canada). To obtain other properties ofthese molecules, the same software was employed to calculate the 2D and3D descriptors. The properties summarized in Table 1 enabled us todivide these molecules into two groups. The first group includes themolecules with more than one ring: HPB—SO₃Na, PG(E01)PhSO₃Na andTA-PhSO₃Na. It should also be mentioned that when the number of aromaticrings on the displacers in this group was increased, these moieties wereadded to the termini of the chains attached to the core ring structure.Hydrophobicity is another significant property of the displacers thatconstitute the first group. The remaining molecules with only the corering form the second group. We can summarize the important properties ofthese homologous series of molecules as follows: According to Table 1,the most hydrophobic displacer and the displacer with the most aromaticrings was HPB—SO₃Na. PG(E01)PSNa₃ and PG(E01)PhSO₃Na are differentbecause the latter has 3 more aromatic rings at the termini as is thecase for TaPSNa₃ and TaPhSO₃Na. ICPSNa₃ was the only displacer withoutany aromatic ring. PGPSNa₃ and TaPSNa₃ were different from each other interms of the type of the aromatic ring they contain, benzene andtriazine, respectively.

TABLE 1 Properties of the displacers LogP # of Molecule MW FCharge¹b_ar² (o/w)³ rings⁴ a_acc⁵ HPB-SO₃Na 1146.9 −6 42 6.736 7B 0 PG(E01)Ph-792.7 −3 24 2.397 4B 6 SO₃Na TA-PhSO₃Na 663.5 −3 24 0.946 3B + 6 1TPG(E01)PSNa₃ 690.6 −3 6 −1.350 1B 6 PG-PSNa₃ 558.5 −3 6 −0.362 1B 3TA-PSNa₃ 561.4 −3 6 −2.804 1T 6 IC-PSNa₃ 561.4 −3 0 −2.855 1T 0¹Fcharge: Formal charge on the molecule ²b_ar: number of aromatic bonds³logP(o/w): octanol/water partition coefficient ⁴B = benzene, T =triazine ⁵a_acc: number of hydrogen-bond acceptor atoms

The linear SMA parameters of the displacer molecules were determined oneach stationary phase, as described in the experimental section, and thevalues are shown in Table 2. The parameters for PG(E01)Ph—SO₃Na couldnot be obtained because the displacer had such a high affinity that itdidn't elute from any of the stationary phases under the experimentalconditions. As shown in the Table, there were significant differences inthe values of these parameters as the stationary phase and/or thedisplacer compound was changed. For example, the characteristic chargeswere slightly higher on the Source 15Q resin, than for the other tworesins which indicates that most of the charged groups on the displacerswere effectively interacting with the charged sites on this stationaryphase. The equilibrium constant (K) values were also slightly higher onSource 15Q resin especially for HPB—SO₃Na and TA-PhSO₃Na, whichindicates that, in general, the displacers were bound more strongly tothe resin. These results demonstrate that the mode of interaction andthe strength of binding can be affected by both displacer and stationaryphase chemistry.

TABLE 2 Linear SMA parameters of the displacers on different stationaryphases ToyoPearl Stationary Phase Q Sepharose HP Source 15Q SuperQ-650SDisplacer v K v K v K HPB-SO3Na 4.11 102.0 4.36 478.0 3.76 54.8TA-PhSO3Na 2.22 29.00 1.9 169.0 1.67 83.7 PG-PSNa3 2.18 1.59 3.04 9.932.54 2.61 TA-PSNa3 2.81 0.416 3.02 1.18 2.69 0.39 PG(E01)PSNa3 2.550.374 2.56 3.12 2.61 0.454 IC-PSNa3 2.54 0.352 3.07 3.21 2.7 0.040

By evaluating the dynamic affinity of these displacer molecules, wecould examine the relative affinities that would be expected under thenon-linear binding conditions found in displacement chromatography.Dynamic affinity plots are log—log plots and thus only significantchanges in the dynamic affinities will be observed. Further, while thevalue of log (K) will affect the general location of the dynamicaffinity lines, only the characteristic charge affects the slope ofthese lines.

The Q Sepharose HP medium is composed of a rigid cross-linked agarose.Agarose is an example of a polysaccharide stationary phase material,which has a high degree of hydrophilicity because of the presence of ahigh density of free OH groups. It is an alternating copolymer of(1–3)-b-D-galactopyranose and (1–4)-3,6-anhydro-a-L-galactopyranose andis hydrophilic. A functional group (quaternary ammonium) is added to thesaccharides through a —CO—NH—CH—O grouping.

It was observed that the molecules belonging to the first group (>/ring)had higher affinities on the Q Sepharose HP stationary phase. The orderfor the first group was: PG(E01)PhSO₃Na>HPB—SO₃Na>TA-PhSO₃Na. Onepossible reason for PG(E01)PhSO₃Na having a very high affinity could bethe longer chains attached to the middle aromatic ring which willpossibly make the molecule more flexible than the other displacers foundin this group because the flexibility will enable it to interact withthe backbone matrix. The second group followed the order ofPG-PSNa₃>TAPSNa₃>PG(EO1)PSNa₃=IC—PSNa₃. Even though they all havesimilar characteristic charges, PG-PSNa₃ has one aromatic ring thatapparently makes it superior to the other molecules in this group.However, having one aromatic group like PG-PSNa₃, PG(E01)PSNa₃ has thelowest affinity in this group. Log P values indicate that PGPSNa₃ (logP=−0.362) is more hydrophobic than PG(E01)PSNa₃ (log P=−1.35), which mayhave caused the difference in their efficacy for the agarose-basedresin. Affinity ranking plots have also shown that PGPSNa₃ binds morestrongly than TAPSNa₃, which is indicative of the higher efficacy ofbenzene rings over triazine rings. It also indicates that even thoughTAPSNa₃ has more hydrogen bonding capability than PGPSNa₃, aromaticitydominates the mode of interaction on the agarose resin. IC—PSNa₃ has thelowest affinity, which clearly shows that having no aromatic rings has asignificant impact on lowering the efficacy of the displacer. Inaddition, the difference in the affinity of the first group and secondgroup of displacers was more pronounced than it was for the otherstationary phases. The SMA parameters indicate that all the moleculeshave a characteristic charge in the range of 2–3 with the exception ofHPB—SO₃Na, which has a characteristic charge of 4.11.

Source 15Q is a hydrophilized PS-DVB resin used for proteinchromatography. FIG. 1 shows the dynamic affinities of the displacersfor the Source 15Q stationary phase. As for the Q Sepharose HPstationary phase, the displacers in the first group have higher affinitythan the displacers in the second group. The order of affinity in thefirst group is: PG(E01)PhSO₃ [not shown]>TAPhSO₃Na>HPB—SO₃Na. Eventhough the dynamic affinity line of TAPhSO₃Na lies above HPBSO₃Na, wehave to make clear that the former displacer has a lower characteristiccharge, which causes the dynamic affinity line to be steeper. However,HPB—SO₃Na has a shallower line (high ν and K), which indicates that thisdisplacer will be more effective over a wide range of operatingconditions. The order of dynamic affinity for the second group wasPGPSNa₃>ICPSNa₃=PG(E01)PSNa₃>TAPSNa₃. Similarly to the previousstationary phase, PGPSNa₃ with one benzene ring has superior affinity tothe other displacers with no aromatic rings or a triazine ring. Ingeneral, the equilibrium constants obtained on this stationary phasewere higher than they were for the other resins, which was indicative ofstronger binding. This result could be due to the aromatic ring contentof this resin (PS/DVB), which will contribute strongly to the π—πinteractions. In contrast to Q Sepharose HP, the affinity differencesbetween the first and second group displacers were not as pronounced forthis stationary phase. As with the previous resin,aromaticity/hydrophobicity was still the dominating secondaryinteraction in increasing the efficacy of displacers on thehydrophilized PS/DVB Source 15Q resin.

ToyoPearl Super Q-650S ion exchangers consist of apoly(methyl)methacrylate backbone bearing quaternary ammonium groups.Dynamic affinities of the displacers were determined for this stationaryphase. As before, the dynamic affinity lines for the first group with ahigh number of aromatic rings lie above the second group of displacers.The order of affinity for the first group of displacers was:PG(E01)PhSO₃Na>TaPhSO₃Na>HPB—SO₃Na. Once again, because of the shallowerdynamic affinity line, HPB—SO₃Na will be more effective over a range ofoperating conditions. The order of dynamic affinity for the second groupof displacers was: PGPSNa₃>TAPSNa₃=PG(E01)PSNa₃>ICPSNa₃. The benzenering was significantly more efficacious than the triazine ring, whichwas true for all of the stationary phases, employed for this study. Itwas demonstrated that aromaticity is very significant in increasing theefficacy of the displacers on the poly(methyl)methacrylate resins.

In addition to obtaining SMA parameters for the various displacers, wealso obtained SMA parameters for a set of proteins on Source 15Qstationary phase. The set consisted of proteins that have been employedcommonly in the literature to demonstrate the efficiency of anionexchange resins for protein purifications. It was clear that all of thedisplacers synthesized for this study had dynamic affinities greaterthan the proteins in this set. This result indicated that the anionexchange displacement experiments that would be carried out using thesedisplacers may be effective for very challenging protein purifications(e.g. HSA and BSA, β-lactoglobulin A and β-lactoglobulin B). As seen inFIG. 2, displacement separation of β-lactoglobulins was successfullycarried out using PGPSNa₃ as a displacer.

In addition, the use of very low concentrations of very high affinitydisplacers, such as HPBSO₃Na, in an anion exchange displacementchromatographic purification may result in very high resolution proteinseparations.

The common trend for all of the resins employed for this study was thatthe displacers with a high degree of aromaticity (increasing number ofaromatic bonds) had very high affinities. For Q Sepharose HP andToyoPearl SuperQ-650S, the displacer without any aromatic bonds(IC—PSNa₃) had the least affinity. Furthermore, the results indicatedthat benzene rings were superior to triazine rings in increasing theaffinity for anion exchange systems. The affinity differences betweenthe first and second group of displacers were much more pronounced on QSepharose HP resin. In all cases, HPSO₃Na with the shallower dynamicaffinity line was shown to be efficacious over a wide range of operatingconditions. As analyzed for the Source I SQ stationary phase, all thedisplacers synthesized in this work can be employed for the purificationof very challenging protein mixtures.

Example 3 High Throughput Screening of Displacers for the Purificationof Oligonucleotides

Materials: Bulk strong anion exchange (quaternary ammonium), Source 30Q(30 μm) was kindly donated by Amersham Biosciences (Uppsala, Sweden).Poros HQ/H (10 μm), 100×4.6 mm I.D. was obtained from PerseptiveBiosystems (Framingham, Mass., USA).

Apparatus: Oligonucleotide analysis was carried out using a Waters 600multisolvent delivery system, a Waters 712 WISP autoinjector and aWaters 484 UV-Vis absorbance detector controlled by a Milleniumchromatography manager (Waters, Milford, Mass., USA).

Procedure: The bulk stationary phase (Source 30Q; 1.8 ml) was firstwashed with deionized water and then the mobile phase buffer, 20 mMNaOH+500 mM NaCl was added and allowed to equilibrate for 2 hours. Aftergravity settling of the stationary phase, the supernatant was removedand 21 ml of 15 mg/ml phosphorothioate anti-sense oligonucleotide in themobile phase buffer was then added and equilibrated in a shaker for 12hours at 23° C. The supernatant was then analyzed by high temperature(70° C.) anion exchange chromatography to determine the oligonucleotideconcentration and the amount adsorbed on the stationary phase wascalculated through a mass balance. The supernatant was then removed and25 μl aliquots of the stationary phase with adsorbed oligonucleotidewere added to separate vials. Aliquots (300 μl) of 10 mM solutions ofeach displacer in the mobile phase buffer were then added to individualvials, and were allowed to equilibrate for 6 hours. After equilibrationwas complete, the stationary phase was allowed to gravity-settle and thesupernatants were removed and analyzed to determine the percentage ofoligonucleotide displaced by each displacer. These experiments werecarried out in duplicate. Results appear in Table 3.

TABLE 3 HTS of Displacers for Oligonucleotides % Oligos DisplacedDisplacer Average Vial 1 Vial 2 Buffer Control 1.9 2.1 1.7 IC-PSNa₃ 42.142.2 42.1 TA-PSNA3 42.3 43.3 41.3 PG(EO1)-PSNa₃ 44.3 42.8 45.8 PG-PSNa₃46.8 46.4 47.2 PG(EO1)-PhSO₃Na 80.4 77.0 83.6

The results indicate that displacers of formula I can be effective inchromatographic separations of oligonucleotides. In particular, thecompound that displaced the highest amount of oligonucleotide wasPG(E01)-PhSO₃, which displaced 80% of the oligonucleotide from thestationary phase material.

1. A method for separating one or more components of a biomoleculemixture by means of an ion exchange chromatographic system operated inthe displacement mode, said method comprising sequentially perfusing thesystem with a first solution comprising the biomolecule mixture, and asecond solution comprising a displacer of structure

wherein Ar is phenyl, naphthalenyl, phenanthrenyl, anthracenyl,perylenyl, pyrenyl, fluorenyl, triazolyl, carbazolyl, indolyl,benzocarbazolyl, acridinyl, phenothiazinyl, or thianthrenyl; X⁴ issulfonate, sulfate, phosphate, carboxylate, tertiary amino or quaternaryamino; Z is CH or N; and n is an integer from 1 to
 6. 2. A methodaccording to claim 1, wherein the displacer is an anion.
 3. A methodaccording to claim 1, wherein the displacer is a cation.
 4. A methodaccording to claim 1, wherein the biomolecule mixture comprises at leastone protein.
 5. A method according to claim 1, wherein the biomoleculemixture comprises at least one oligonucleotide.
 6. A method according toclaim 1, additionally comprising equilibrating the system with a carriersolution before sequentially perfusing the system with the firstsolution.
 7. A method according to claim 1, additionally comprisingregenerating the system after perfusing the system with the secondsolution.
 8. A method according to claim 1, wherein Ar is phenyl.
 9. Amethod according to claim 1, wherein Ar is triazolyl.
 10. A methodaccording to claim 1, wherein the displacer is